Herpesviruses, such as herpes simplex virus-1 (HSV-1), rely on the ability to egress, in a process termed budding, where progeny virions are released from the cell and spread to uninfected tissues and hosts. HSV-1 causes lifelong infections of the host resulting in diseases such as blindness, encephalitis, keratitis, and cancers and currently there is no cure or vaccine. High rates of new infections, and reactivation of latent infections, stem from the ability of the virus to egress. Although egress is not unique to herpesviruses, unusually, herpesviruses bud twice, rather than only once as found for most other viruses. Specifically, herpesviruses first bud at the inner nuclear membrane (INM) where viral capsids form nascent buds that pinch off into the perinuclear space (scission). These buds then fuse with the outer nuclear membrane releasing capsids into the cytosol. Capsids subsequently bud again at the cytoplasmic membranes derived from early endosomes or the trans-Golgi network. The first HSV-1 egress event, nuclear budding, relies on the conserved nuclear egress complex (NEC), a heterodimer composed of two viral proteins, UL31 and UL34, both necessary for the exit of viral capsids from the nucleus. A major barrier in the understanding of how this complex mediates budding is the lack of knowledge regarding the conformational changes the NEC undergoes to achieve budding, how the complex interacts with membranes, and how this process is regulated during viral infection. A deep knowledge of the mechanism of NEC budding may hold the key to curing this disease by blocking the spread of future infections. Therefore, this proposal is based on the central hypothesis that NEC- mediated budding is a dynamic, complex process that is regulated by multiple factors during infection, including the NEC itself. Three specific aims underpinned by this central hypothesis form a multipronged approach combining several biophysical techniques to address the significant gaps in our knowledge regarding the NEC budding mechanism. First, the effects on NEC lattice formation of both non-budding NEC mutants and an NEC mutant that restores budding in non-budding mutants will be investigated with cryoelectron microscopy/tomography (cryoEM/T) to analyze NEC conformational changes. A comparison of these lattices to the wild-type NEC lattice will provide details as to how specific residues contribute to NEC lattice formation and enable budding. Secondly, the role of membrane-interacting regions of the NEC will be probed using mutagenesis, confocal microscopy budding quantification, and cryoET. Finally, the regulation of NEC budding will be investigated by determining how another HSV-1 protein binds to the NEC and how this binding has an effect on NEC budding efficiency utilizing small angle X-ray scattering (SAXS), mutagenesis, and cryoET. All together, these experiments will add to the understanding of nuclear budding in general and ultimately aid in the development of novel therapeutic agents to prevent the spread of future HSV-1 infections.